Biogel

ABSTRACT

A biogel, and kits, agents, and methods for formation of the biogel are described. The biogel can be used for a variety of applications, including haemostasis, wound sealing, tissue engineering or localised drug delivery.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 430160_403 D1_SEQUENCE_LISTING.txt. The textfile is 5.4 KB, was created on Apr. 12, 2016, and is being submittedelectronically via EFS-Web.

This invention relates to a biogel, and to kits, agents, and methods forformation of the biogel. In a preferred aspect the biogel is a tissueadhesive. The biogel or tissue adhesive can be used for a variety ofapplications, including haemostasis, wound sealing, tissue engineeringor localised drug delivery.

During the clotting process, fibrinogen is converted to fibrin bythrombin. Fibrinogen comprises two sets of three different chains (α, β,and γ), linked to each other by disulfide bonds. Together these chainsform a central globular domain (the E domain) connected to two distalglobular domains (the D domains). Thrombin cleaves four arginine-glycinepeptide bonds in the central E domain of fibrinogen to release an Apeptide from each of the two α chains, and a B peptide from each of thetwo β chains. The A and B peptides are termed fibrinopeptides. Afibrinogen molecule devoid of these fibrinopeptides is called a fibrinmonomer. Fibrin monomers spontaneously assemble into ordered fibrousarrays called fibrin. Fibrin is stabilised by the formation of covalentcross-links between the side chains of different molecules in the fibrinfibre. Peptide bonds are formed between specific glutamine and lysineside chains in a transamidation reaction that is catalysed by FactorXIIIa.

Once activated, platelets also form an essential part of a blood clot.Platelets adhere to an exposed wound surface and become activated. Theplatelet membrane glycoprotein GPIIb/IIIa undergoes a change inconformation, which allows it to bind fibrinogen. Fibrinogen can bind tomore than one platelet, and so platelets aggregate together. Plateletaggregates form the basic architecture of the clot, formed within a meshof fibrin.

Fibrin tissue adhesive (FTA) is the name given to products formed bymimicking the last step of the coagulation cascade to form a fibrinclot. Commercially available FTA kits rapidly produce strong,biodegradable gels that are used for haemostasis, drug delivery, and assurgical glues, and tissue sealants. Fibrinogen, Factor XIII, thrombin,and calcium ions are typically delivered via a syringe device thatseparates fibrinogen and Factor XIII from calcium ions and thrombinduring storage. Mixing of the components during discharge from thesyringe results in thrombinolysis of fibrinogen to create fibrin, whichself-assembles into a gel that is later cross-linked by calciumion-activated Factor XIII. However, many FTAs utilise bovine thrombin,which can induce anaphylactic and autoimmune responses in patients.

Zhang et al. (Bioconjugate Chem. 2002 (13): 640-646) describe formationof fibrinogen-based hydrogels by photoactivated release of calcium ionsfrom liposomes, and subsequent activation of transglutaminase-catalyzedcross-linking of fibrinogen. However, formation of these hydrogels iscomplicated, and specialized formulation of liposome and Factor XIII isrequired.

Hidas et al. (Urology 67(4), 2006: 697-700) describe use of albuminglutaraldehyde tissue adhesive in sutureless nephron-sparing surgery.Bovine serum albumin and glutaraldehyde were admixed. Glutaraldehydeexposure causes the lysine molecules of the bovine serum albumin,extracellular matrix proteins, and cell surfaces to bind to each other,creating a strong covalent bond. A disadvantage of this adhesive,however, is that glutaraldehyde is toxic, and there is a risk thatbovine serum albumin can induce an allergic reaction in patients.

There is, therefore, a need to provide gel or tissue adhesive which doesnot require use of toxic agents, which minimizes the risk of allergicreaction, and which is simple to produce from components which canreadily be stored in a stable condition.

According to the invention there is provided a biogel kit (i.e. a kitfor formation of a biogel), which comprises: a plurality of carriers, aplurality of fibrinogen binding moieties being immobilised to eachcarrier; and fibrinogen, wherein each molecule of fibrinogen can bind atleast two fibrinogen binding moieties.

The term “biogel” is used herein to include a gel comprising one or morecomponents that are natural or recombinant biological molecules (orchemically synthesised biological molecules), or that are derived frombiological molecules (for example derivatives that retain one or morefunctions of a biological molecule).

A biogel can be formed by contacting the fibrinogen and the carriers.Because a plurality of fibrinogen binding moieties are immobilised toeach carrier, and because each fibrinogen molecule can bind at least twoof the fibrinogen binding moieties, the fibrinogen molecules becomelinked together via the carriers. Non-covalent bonds are formed betweenthe fibrinogen molecules and the fibrinogen binding moieties.

Accordingly, there is also provided according to the invention a methodof forming a biogel, which comprises contacting fibrinogen moleculeswith a plurality of carriers, wherein each carrier has a plurality offibrinogen binding moieties immobilised to the carrier, and eachmolecule of fibrinogen can bind at least two fibrinogen bindingmoieties, so that the fibrinogen molecules become linked together viathe carriers by formation of non-covalent bonds between the fibrinogenbinding moieties and the fibrinogen molecules.

There is further provided according to the invention a biogel whichcomprises fibrinogen molecules and a plurality of carriers, wherein eachcarrier has a plurality of fibrinogen binding moieties immobilised tothe carrier, and each molecule of fibrinogen is bound to at least twofibrinogen binding moieties, so that the fibrinogen molecules are linkedtogether via the carriers by non-covalent bonds between the fibrinogenbinding moieties and the fibrinogen molecules.

Instead of fibrinogen binding moieties, the carriers may have aplurality of fibrinogen binding precursors immobilised to each carrier,wherein each fibrinogen binding precursor can be converted to afibrinogen binding moiety. To form a biogel using such carriers, it isnecessary to convert the fibrinogen binding precursors to fibrinogenbinding moieties so that the fibrinogen binding moieties can then bindto the fibrinogen molecules.

Accordingly there is also provided according to the invention a biogelkit (i.e. a kit for formation of a biogel), which comprises: a pluralityof carriers, a plurality of fibrinogen binding precursors beingimmobilised to each carrier, wherein each fibrinogen binding precursorcan be converted to a fibrinogen binding moiety; and fibrinogen, whereineach molecule of fibrinogen can bind at least two fibrinogen bindingmoieties.

There is further provided according to the invention a method of forminga biogel, which comprises: providing a plurality of carriers, eachcarrier comprising a plurality of fibrinogen binding precursorsimmobilised to the carrier; converting the fibrinogen binding precursorsto fibrinogen binding moieties; and contacting fibrinogen molecules withthe fibrinogen binding moieties, wherein each molecule of fibrinogen canbind at least two fibrinogen binding moieties, so that the fibrinogenmolecules become linked together via the carriers by formation ofnon-covalent bonds between the fibrinogen binding moieties and thefibrinogen molecules.

A biogel of the invention need not be capable of adhering to a tissuesubstrate. However, in preferred aspects a biogel of the invention is atissue adhesive. The term “tissue adhesive” is used herein to mean asubstance that can adhere to a tissue substrate, for example skin, or amucosal surface. Biogels or tissue adhesives of the invention may beused in haemostasis, as sealants, for tissue engineering (for example asa support), or for localised drug delivery.

The carrier may be a soluble or insoluble carrier, but is not aplatelet. The carrier should be suitable for topical administration to atissue site of a subject, for example a bleeding wound site, or amucosal site. Soluble carrier may be suitable for intravenous ratherthan topical administration. The carrier may comprise a soluble orinsoluble protein, a therapeutic drug, a polymer (for example abiocompatible polymer, such as polyethylene glycol), or a combination ofany of these.

Examples of protein carriers are an enzyme or a protein which is not anenzyme, such as human serum albumin.

An insoluble carrier may be a microparticle (including a solid, hollow,or porous microparticle, preferably a substantially sphericalmicroparticle). The microparticle may be formed of any suitablesubstance, for example cross-linked protein. A suitable protein isalbumin (serum-derived or recombinant, human or non-human in sequence).Microparticles suitable for use as insoluble carriers in the presentinvention may be formed by spray drying human serum albumin using wellknown spray-drying technology, for example as in WO 92/18164.

Alternatives to use of microparticles as carriers include liposomes,synthetic polymer particles (such as polylactic acid, polyglycolic acidand poly(lactic/glycolic) acid), or cell membrane fragments.

The term “fibrinogen” is used herein to include natural fibrinogen,recombinant fibrinogen, or a derivative of fibrinogen that can beconverted by thrombin to form fibrin (for example, natural orrecombinant fibrin monomer, or a derivative of fibrin monomer that mayor may not be capable of spontaneous assembly). The fibrinogen should beable to bind at least two fibrinogen binding moieties. The fibrinogenmay be obtained from any source, and from any species (including bovinefibrinogen), but is preferably human fibrinogen. Human fibrinogen may beobtained from autologous or donor blood. Autologous fibrinogen ispreferred because this reduces the risk of infection when biogel (oradhesive) of the invention is administered to a subject.

Preferably the fibrinogen binding moiety binds to fibrinogen with adissociation constant (K_(D)) of between 10⁻⁹ to 10⁻⁶ M, for examplearound 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,200, 250, 300, 350, 400, or more nM. A K_(D) of around 100 nM ispreferred. The dissociation constant can be measured at equilibrium. Forexample, radio-labelled fibrinogen of known concentration can beincubated with microspheres to which the fibrinogen binding moiety hasbeen cross-linked. Typically 5 μM peptide is cross-linked to 1 gmmicrospheres, or 15-40 μmoles of peptide is cross-linked to 1 gm ofmicrospheres. The peptide-linked microspheres are diluted to 0.5 mg/ml,and incubated in isotonic buffer at pH 7.4 (for example 0.01M Hepesbuffer containing 0.15M NaCl) with radio labelled fibrinogen atconcentrations of between 0.05 and 0.5 mg/ml for up to 1 hr at 20° C.The fibrinogen bound to the fibrinogen binding moiety on themicrospheres can be separated from the free fibrinogen by centrifugationand the amount of free and bound fibrinogen measured. The dissociationconstant can then be calculated by Scatchard analysis by plottingconcentration of bound fibrinogen against the ratio of theconcentrations of bound:free fibrinogen, where the slope of the curverepresents K_(D).

In some embodiments of the invention it is preferred that the fibrinogenbinding moiety binds selectively to fibrinogen. In other embodiments itis preferred that the fibrinogen binding moiety can bind to fibrinogen,and separately to fibrin monomer and/or fibrin. Binding to fibrinogenand fibrin monomer and/or fibrin is preferably selective.

Preferably the fibrinogen binding moiety is a fibrinogen binding peptideor peptide analogue. Any suitable fibrinogen binding peptide may beused. For example, the peptide may be capable of binding to a region offibrinogen that is naturally bound to fibrin or by the platelet membraneglycoproteins GPIIb-IIIa. Fibrin binding to fibrinogen is discussed inMosesson et al. 2001, Ann. N.Y. Acad. Sci., 936, 11-30. Binding ofGPIIb-IIIa to fibrinogen is discussed in Bennett, 2001, Annals of NYAcad. Sci., 936, 340-354.

The peptide may be capable of binding to the carboxy- and/oramino-terminal domain of the α-chain of fibrinogen. In particular, thepeptide may be capable of binding to an RGD-containing motif in one orboth of these domains (such as RGDF (SEQ ID NO: 1) at amino acids 95-98,or RGDS (SEQ ID NO: 2) at amino acids 572-575). The peptide may becapable of binding to the carboxy-terminal domain of the γ-chain offibrinogen, preferably to the final 12 amino acids of this domain(sequence HHLGGAKQAGDV (SEQ ID NO: 3)). The peptide may be capable ofbinding to the D-domain of the γ-chain of fibrinogen, such as theβ-chain segment of the D-domain.

The fibrinogen binding peptide may comprise a sequence derived from afibrinogen-binding region of GPIIb or GPIIIa. For example, the peptidemay comprise the sequence AVTDVNGDGRHDLLVGAPLYM (SEQ ID NO: 4) whichcorresponds to the sequence of amino acid residues 294-314 of GPIIb, ora fragment or derivative thereof that retains fibrinogen bindingactivity. Fragments known to retain fibrinogen binding activity areTDVNGDGRHDL (296-306) (SEQ ID NO: 5), GDGRHDLLVGAPL (300-312) (SEQ IDNO: 6), and GAPL (SEQ ID NO: 7). Suitable derivatives of TDVNGDGRHDLinclude: T(D,E)VNG(D,E)GRH(D,E)L (SEQ ID NO: 8); TD(V,L)NGDGRHDL (SEQ IDNO: 9); TDV(N,Q)GDGRHDL (SEQ ID NO: 10); TDVNGDG(R,K)HDL (SEQ ID NO:11).

The fibrinogen binding peptide may comprise the sequence of residues95-223 of GPIIIa, or a fragment or derivative thereof that retainsfibrinogen binding activity. For example, residues 211-222, comprisingthe sequence SVSRNRDAPEGG (SEQ ID NO: 12), are thought to be animportant fibrinogen-binding domain in GPIIIa. Other suitable regions ofGPIIIa include residues 109-171 and 164-202.

The fibrinogen binding peptide may comprise the sequence of residueswhich becomes exposed on fibrinogen by the action of thombin, and whichbinds fibrinogen as the first step in the polymerisation reaction toproduce fibrin. Thrombin cleaves peptides (releasing fibrinopeptides Aand B) from the N terminals of the α and β chains of fibrinogen toexpose the sequences NH₂-GPR- (SEQ ID NO: 13) and NH₂-GHR- (SEQ ID NO:14) respectively. A preferred example of a fibrinogen binding peptidetherefore comprises the amino acid sequence NH₂-G(P,H)RX- (SEQ ID NO:15) at its amino terminal end, where X is any amino acid, and (P,H)means that either proline or histidine is present at that position.Preferably the peptide comprises the sequence NH₂-GPRP- (SEQ ID NO: 16)at its amino terminal end.

Preferably the fibrinogen binding peptide is 4-30, more preferably 4-10,amino acid residues in length.

The fibrinogen binding precursor should not bind to fibrinogen such thatfibrinogen molecules become linked together via carriers havingimmobilised fibrinogen binding precursors when the carriers are incontact with fibrinogen. Preferably the dissociation constant of thefibrinogen binding precursor for fibrinogen is greater than 1×10⁶M. Thefibrinogen binding precursor may be a peptide or a peptide analogue, butis preferably a peptide. The fibrinogen binding precursor should not befibrinogen nor comprise fibrinogen.

In preferred embodiments of the invention the fibrinogen bindingprecursor comprises a fibrinogen binding peptide joined at its aminoterminal end to a blocking component (preferably a peptide) that blocksor inhibits (i.e. reduces) binding of fibrinogen to the fibrinogenbinding peptide. Cleavage of the fibrinogen binding precursor by aconverting agent (preferably a coagulation factor such as thrombin)exposes the fibrinogen binding peptide bound to the carrier, therebyconverting the fibrinogen binding precursor to a fibrinogen bindingmoiety. In such embodiments, the blocking component blocks or inhibitsthe ability of the fibrinogen binding peptide to bind fibrinogen untilcleavage occurs. Preferably the blocking component is a peptide of 1-30amino acid residues in length.

It will be appreciated that in such embodiments the fibrinogen bindingprecursor should comprise a cleavage site that is recognisedspecifically by the converting agent and which is located between thefibrinogen binding peptide and the blocking component. Thrombin is apreferred converting agent. However, other serine proteases orcoagulation factors may be used to cleave the fibrinogen bindingprecursor. Thrombin is known to cleave peptide bonds carboxy-terminal toarginine residues, and commonly between arginine and glycine residues.

In particularly preferred embodiments of the invention the fibrinogenbinding precursor is a peptide which comprises the amino acid sequenceNH₂-ZYXR/GPRP- (SEQ ID NO: 17) at its amino terminal end, where “/”represents a thrombin cleavage site, and X is any amino acid, but ispreferably proline, Y is any amino acid, but is preferably aspartic acidor alanine, and Z is at least one amino acid that is preferably leucineor proline. Examples are: NH₂-LVPR/GPRP- (SEQ ID NO: 18), NH₂-ADPR/GPRP-(SEQ ID NO: 19), NH₂-LDPR/GPRP- (SEQ ID NO: 20), or NH₂-LVPR/GPRV- (SEQID NO: 21).

The fibrinogen binding moieties or precursors can be bound to thecarrier by any suitable means, but are typically covalently bound.Examples of preferred covalent bonds are a disulphide bond, a thioetherbond, or an amide bond. A suitable covalent bond can be formed when thefibrinogen binding moieties or precursors are peptides which comprise acysteine and the carrier comprises a thiol reactive group. This allowsthe peptide to be bound to the carrier by linking the —SH group of thecysteine to the thiol-reactive group on the carrier. Preferably aterminal cysteine group is incorporated in the fibrinogen bindingpeptide or precursor peptide to crosslink the peptide with athiol-reactive group on the carrier. Alternatively, a covalent bond maybe formed when the fibrinogen binding moiety or precursor is a peptidewhich comprises a maleimide group (preferably at its carboxy terminus,for example attached to a carboxy-terminal Lysine of the peptide), andthe carrier comprises a sulphydryl group. The peptide may then be boundto the carrier by reacting the maleimide group of the peptide with thesulphydryl group of the carrier.

Typically, a spacer will be required between the fibrinogen bindingmoieties or precursors and the carrier to ensure that thefibrinogen-binding activity of the fibrinogen binding moieties (ifnecessary once converted from the fibrinogen binding precursors) is notadversely affected by the carrier. Suitable spacers are peptides, or nonpeptides such as polyethylene glycol.

Where the fibrinogen binding moieties or precursors are peptides whichcomprise a fibrinogen binding peptide, and the moieties or precursorsare bound to the insoluble carrier by a terminal amino acid residue, aspacer sequence is preferably present between the terminal amino acidresidue and the fibrinogen binding peptide of the moieties orprecursors. The spacer sequence may, for example, be from 1-20,preferably 5-20, amino acid residues in length. The spacer sequenceGGGGGG (SEQ ID NO: 22) or GGGGG (SEQ ID NO: 23) is preferred.

It will be appreciated that the number of fibrinogen binding moieties orprecursors per carrier and the relative amounts of carrier (with aplurality of fibrinogen binding moieties or precursors per carrier) andfibrinogen required for optimal biogel (or tissue adhesive) formationmay vary with different preparations of carrier and fibrinogen.Accordingly it may be necessary or desirable to test each new batch ofcarrier or fibrinogen to determine the optimum relative amounts ofcarrier and fibrinogen to use for biogel formation.

Preferably each carrier has on average at least five fibrinogen bindingmoieties or precursors per carrier. In theory there is no upper limit tothe number of fibrinogen binding moieties or precursors per carrier. Theoptimum number is likely to depend on many factors, such as the natureof the carrier, and the number of reactive groups on each carrier forattaching the fibrinogen binding moities or precursors. However, it ispreferred that each carrier has on average up to 100 fibrinogen bindingmoieties or precursors per carrier. A preferred range is 10-20fibrinogen binding moieties or precursors per carrier.

Preferably the amount of fibrinogen used is such that there is at leastone quarter (preferably at least one half) of the number of moles offibrinogen present compared to the number of moles of fibrinogen bindingmoiety or precursor. Preferably the number of moles of fibrinogenrelative to the number of moles of fibrinogen binding moiety orprecursor is in the range 1:4 to 4:1.

Dynamic oscillatory measurements may be used to evaluate theviscoelastic properties of a biogel formed according to the invention.The storage (G′) and loss modulus (G″) in viscoelastic solids measurethe stored energy, representing the elastic portion, and the energydissipated as heat, representing the viscous portion. Tan delta is theratio of the loss modulus (G″) to the storage modulus (G′). It istherefore a quantification of the elastic and viscous contributions,where a value above 1 is indicative of liquid-like viscous behaviour,and a value below 1 signifies elastic behaviour. Biogels of theinvention preferably have a tan delta value of less than 1. Thisprovides an indication that the components of the gel are crosslinked.Tan delta may be determined at a frequency of 1 Hz and a constant strainof 1%. Suitable methods for measurement of tan delta are described inmore detail in the examples below.

The components of a kit of the invention may be stored separately fromeach other, or some or all of the components of the kit may be storedtogether provided that components stored together will not react witheach other. For embodiments of the invention in which the kit comprisescarriers with immobilised fibrinogen binding moieties, it will beappreciated that the fibrinogen should be stored separately from thecarriers so that it does not react with the carriers. An importantadvantage of kits of the invention that comprise fibrinogen and carrierswith immobilised fibrinogen binding precursors that do not bindfibrinogen is that the carriers and fibrinogen can be stored together.

Components of a kit of the invention may be stored separately from eachother in a device (for example a syringe) for delivery of the componentsto a tissue or other site. The device may be arranged so that thecomponents contact each other as they are delivered at the tissue orother site.

A kit of the invention may include instructions on how to use thecomponents of the kit to produce a biogel or tissue adhesive.

Kits of the invention have the advantages that no toxic agents arerequired, biogel (or tissue adhesive) is simple to produce using thecomponents of the kits, and the components can readily be stored in astable condition. It will also be appreciated that there is norequirement for thrombin (or other enzymes) to be present for formationof a biogel or tissue adhesive using a kit of the invention. Kits of theinvention which do not include thrombin are particularly advantageousbecause the risk of allergic reaction to a biogel or tissue adhesiveformed using such kits is reduced compared to biogel or tissue adhesiveformed using exogenous thrombin. A further advantage of kits of theinvention that do not include thrombin (or other enzymes) is that thereis no requirement to store the components of the kit under conditionsthat preserve enzyme activity.

According to some embodiments of the invention, the fibrinogen bindingprecursors can be converted to fibrinogen binding moieties by aconverting agent that is present at a site to which carriers havingimmobilised fibrinogen binding precursors are administered. This has theadvantage that there is no requirement for the converting agent to bepart of the kit. Consequently, there is no requirement to store thecomponents of the kit under conditions that preserve activity of theconverting agent.

Preferably the converting agent is a wound site agent. The term “woundsite agent” is used herein to mean an agent which is present at a woundsite. In a preferred embodiment the wound site agent is a coagulationfactor. Examples of suitable coagulation factors include thrombin,Factor Vila, Factor Xa, or Factor XIa. Preferably the coagulation factoris thrombin.

According to other embodiments, a kit of the invention may furthercomprise a converting agent for converting the fibrinogen bindingprecursors to fibrinogen binding moieties. The converting agent shouldbe separate from the carrier. In such embodiments the converting agentmay be an agent that is not expected to be present at a site to whichthe carriers are to be administered. Alternatively the converting agentmay be an agent that is present at a site to which the carriers areadministered. The converting agent may be a coagulation factor, such asthrombin, Factor Vila, Factor Xa, or Factor XIa.

A kit of the invention comprising carriers with immobilised fibrinogenbinding moieties may further comprise a coagulation factor. A kit of theinvention comprising fibrinogen binding precursors that can be convertedto fibrinogen binding moieties by a coagulation factor may comprise acoagulation factor which does not convert the fibrinogen bindingprecursors to fibrinogen binding moieties. In such embodiments thecoagulation factor (that does not convert the fibrinogen bindingprecursors) may be provided as a separate component of the kit, or withthe carrier and/or fibrinogen. The coagulation factor may be immobilisedto the carrier, for example coupled to each fibrinogen bindingprecursor.

Where a kit of the invention comprises a coagulation factor (for examplethrombin), this is preferably a coagulation factor which is of humansequence, rather than non-human (such as a bovine coagulation factor) toreduce the risk of allergic reaction to the coagulation factor.

A biogel or tissue adhesive of the invention may be formed before beingadministered to a tissue site, or in situ at a tissue site, for exampleat a wound site. Biogel or tissue adhesive at a tissue site preferablycomprises topically administered carrier.

It will be appreciated that thrombin may be present at a tissue site atwhich a biogel or tissue adhesive of the invention is administered orformed. For example, if the tissue site is a bleeding wound site, hostthrombin is likely to be present at the wound site. Of course, thrombinmay alternatively or additionally be present if it is provided with akit of the invention.

Use of fibrinogen binding moieties that are able to bind to fibrinogenand separately to fibrin monomer and/or fibrin may be advantageous ifbiogel or adhesive of the invention is formed in the presence ofthrombin, or comes into contact with thrombin once it is formed. Ifthrombin is present with fibrinogen and carriers having immobilisedfibrinogen binding moieties, the thrombin will convert at least some ofthe fibrinogen to fibrin monomer. It will be appreciated that underthese conditions, it is preferred that the fibrinogen binding moietiesalso bind to fibrin monomers so that the fibrin monomers formed bythrombin can be linked together by the carriers.

If biogel or adhesive of the invention comes into contact with thrombinonce it is formed, it may be advantageous if the fibrinogen bound to thefibrinogen binding moieties is able to be converted to fibrin monomerand the fibrinogen binding moieties remain bound to the fibrin monomer.Under these circumstances non-covalent bonds formed between fibrinogenand the fibrinogen binding moieties will not be disrupted by conversionof fibrinogen to fibrin monomer. It may also be advantageous if at leastsome of the fibrin monomers bound to the fibrinogen binding moieties areable to assemble to form fibrin whilst remaining bound to the fibrinogenbinding moieties. Under these circumstances the biogel or tissueadhesive may be strengthened by the formation of fibrin.

If thrombin is present (for example, from a kit of the invention, orhost thrombin at a wound site) it is preferred that the carriers andfibrinogen are contacted with each other before being contacted withthrombin, or that the carriers, fibrinogen and thrombin are contactedwith each other at substantially the same time.

Whilst it is possible that a biogel or tissue adhesive may be formed ifthrombin and fibrinogen are contacted with each other before contactwith the carriers, this is expected to be of less use than a biogel ortissue adhesive formed by contacting the carriers and fibrinogen witheach other before thrombin, or by contacting the carriers, fibrinogenand thrombin with each other at substantially the same time. This isbecause the thrombin will be expected to convert the fibrinogenmolecules to fibrin monomers which will then aggregate to form insolublefibrin before contact with the carriers. However, it may in somecircumstances be appropriate to form a biogel or tissue adhesive bycontacting the thrombin and fibrinogen before the carriers, for exampleif the thrombin is inactive until or after contact with the carriers.

There is also provided according to the invention a method of forming abiogel, which comprises contacting a plurality of carriers withfibrinogen molecules and thrombin, wherein a plurality of fibrinogenbinding moieties are immobilised to each carrier, and each fibrinogenmolecule can bind at least two fibrinogen binding moieties, to form abiogel in which fibrin monomers are linked together via the carriers bynon-covalent bonds between the fibrinogen binding moieties and thefibrin monomers.

The biogel may or may not comprise fibrin. Accordingly, the fibrinmonomers may be part of fibrin in the biogel, or the fibrin monomers maynot be part of fibrin in the biogel.

There is also provided according to the invention a biogel whichcomprises fibrin monomers and a plurality of carriers, each carrierhaving a plurality of fibrinogen binding moieties immobilised to thecarrier, wherein the fibrin monomers are linked together via thecarriers by non-covalent bonds between the fibrinogen binding moietiesand the fibrin monomers.

There is further provided according to the invention a biogel whichcomprises fibrin and a plurality of carriers, each carrier having aplurality of fibrinogen binding moieties immobilised to the carrier,wherein fibrin monomers of the fibrin are linked together via thecarriers by non-covalent bonds between the fibrinogen binding moietiesand the fibrin monomers.

In a preferred aspect the biogel is a tissue adhesive.

A kit of the invention may comprise Factor XIII, and optionally calciumions and thrombin for activation of Factor XIII to Factor XIIIa (if so,the calcium ions and thrombin should be separate from the Factor XIII).Alternatively or additionally, Factor XIIIa may be present at a tissuesite at which a biogel or tissue adhesive of the invention isadministered or formed. For example, if the tissue site is a bleedingwound site, host Factor XIIIa is likely to be present at the wound site.

If Factor XIIIa is contacted with a biogel or tissue adhesive of theinvention which comprises fibrin, the biogel or tissue adhesive may befurther strengthened by reaction of Factor XIIIa with the fibrin tocovalently cross-link the fibrin.

There is also provided according to the invention a method of forming abiogel, which comprises: contacting a plurality of carriers withfibrinogen molecules and thrombin, wherein a plurality of fibrinogenbinding moieties are immobilised to each carrier, and each fibrinogenmolecule can bind at least two fibrinogen binding moieties, to form abiogel comprising fibrin in which fibrin monomers of the fibrin arelinked together via the carriers by non-covalent bonds between thefibrinogen binding moieties and the fibrin monomers; and contacting thebiogel with Factor XIIIa so that fibrin monomers of the fibrin becomecovalently linked together by peptide bonds.

There is further provided according to the invention a biogel whichcomprises fibrin and a plurality of carriers, each carrier having aplurality of fibrinogen binding moieties immobilised to the carrier,wherein fibrin monomers of the fibrin are covalently linked together bypeptide bonds and fibrin monomers of the fibrin are linked together viathe carriers by non-covalent bonds between the fibrinogen bindingmoieties and the fibrin monomers.

In a preferred aspect the biogel is a tissue adhesive.

A kit of the invention may further comprise a promoter of wound healing.Suitable examples include growth factors, such as platelet-derivedgrowth factor. The promoter may be a separate component of the kit, orimmobilised to the carrier.

A kit of the invention may further comprise an antimicrobial agent, forexample an antibiotic. The antimicrobial agent may be a separatecomponent of the kit, or immobilised to the carrier.

It will be appreciated that if carriers having a plurality of fibrinogenbinding moieties immobilised to each carrier are administered to atissue site at which host fibrinogen is present (for example, at ableeding wound site) the carriers will react with the host's fibrinogento form a biogel or tissue adhesive in situ at the tissue site.

Similarly, if carriers having a plurality of fibrinogen bindingprecursors immobilised to each carrier are administered to a tissue siteat which a converting agent for converting the fibrinogen bindingprecursors to fibrinogen binding moieties, and host fibrinogen arepresent (for example, at a bleeding wound site) the fibrinogen bindingprecursors will be converted to fibrinogen binding moieties, and thecarriers will then react with the host's fibrinogen to form a biogel ortissue adhesive in situ at the tissue site. For example, the fibrinogenbinding precursors may be converted to fibrinogen binding moieties byhost thrombin, or another coagulation factor.

Accordingly, there is further provided according to the invention abiogel which comprises topically administered carriers, each carrierhaving a plurality of fibrinogen binding moieties immobilised to thecarrier, and endogenous fibrinogen, wherein each molecule of endogenousfibrinogen is bound to at least two fibrinogen binding moieties, so thatthe fibrinogen molecules are linked together via the carriers bynon-covalent bonds between the fibrinogen binding moieties and theendogenous fibrinogen molecules.

The term “endogenous fibrinogen” is used herein to mean that thefibrinogen is host fibrinogen, which is present at a site to which thecarriers are administered. Typically the host fibrinogen will be presentbecause blood of the host is present at the wound site.

There is also provided according to the invention use of a carrier forformation of a biogel, the carrier having a plurality of fibrinogenbinding moieties or fibrinogen binding precursors immobilised to thecarrier.

The biogel is preferably a tissue adhesive.

The carrier may be used for formation of a biogel or tissue adhesive forhaemostasis, as a sealant, for localised drug delivery, or for tissueengineering.

Administration of a carrier to form a biogel or tissue adhesive has theadvantage that the risk of host allergic reaction is minimised becausethere is no requirement for exogenous fibrinogen or thrombin, no toxicagents are required, and the carrier can readily be stored in a stablecondition.

There is also provided according to the invention a method ofcontrolling bleeding at a site at which host fibrinogen is present,which comprises topically administering a plurality of carriers to thesite, wherein each carrier has a plurality of fibrinogen bindingmoieties immobilised to the carrier, and host fibrinogen molecules atthe site can bind at least two of the fibrinogen binding moieties.

There is further provided according to the invention a method ofcontrolling bleeding at a site at which host fibrinogen and coagulationfactor is present, which comprises topically administering a pluralityof carriers to the site, wherein each carrier has a plurality offibrinogen binding precursors immobilised to the carrier and thefibrinogen binding precursors can be converted to fibrinogen bindingmoieties by host coagulation factor, and host fibrinogen molecules atthe site can bind at least two of the fibrinogen binding moieties.

There is further provided according to the invention use of a pluralityof carriers in the manufacture of a medicament (e.g. a biogel or tissueadhesive) for controlling bleeding, or for treating or sealing a wound,wherein each carrier has a plurality of fibrinogen binding moieties orfibrinogen binding precursors immobilised to the carrier.

There is also provided according to the invention use of a plurality ofcarriers, each carrier having a plurality of fibrinogen binding moietiesor fibrinogen binding precursors immobilised to the carrier, forformation of a biogel.

There is further provided according to the invention a plurality ofcarriers for use (e.g. as a biogel or tissue adhesive) in controllingbleeding, or for treating or sealing a wound, wherein each carrier has aplurality of fibrinogen binding moieties or fibrinogen bindingprecursors immobilised to the carrier.

The carriers may be insoluble or soluble. Carriers may be administeredtopically.

If the carrier is a soluble carrier, a preferred topical formulation ofthe carrier having immobilised fibrinogen binding moieties or fibrinogenbinding precursors is a liquid formulation, preferably in an isotonicbuffer at physiological pH.

If the carrier is an insoluble carrier, a preferred topical formulationof the carrier having immobilised fibrinogen binding moieties orfibrinogen binding precursors is in the form of a powder that can besprayed, for example, onto a tissue site.

Powdered carrier may be formed using any suitable method, includingmethods comprising spray drying, or lyophilising a suspension of carrierhaving a plurality of fibrinogen binding moieties or fibrinogen bindingprecursors immobilised to each carrier (for example suspended in anisotonic buffer at physiological pH). Preferably spray-drying is usedsince this can be a more rapid and easily scaled method of drying thanlyophilisation. Suitable spray-drying methods are described in WO92/18164.

According to the invention there is provided an agent for formation of abiogel, the agent comprising soluble carrier, wherein a plurality offibrinogen binding moieties or fibrinogen binding precursors areimmobilised to each carrier.

There is also provided according to the invention an agent for formationof a biogel, the agent comprising insoluble carrier having a pluralityof fibrinogen binding moieties or fibrinogen binding precursorsimmobilised to each carrier, wherein the agent is in the form of apowder formed other than by lyophilisation.

There is also provided according to the invention a method of forming apowdered agent, the agent comprising insoluble carrier having aplurality of fibrinogen binding moieties or fibrinogen bindingprecursors immobilised to each carrier, wherein the method comprisesspray-drying a suspension of the agent.

Preferably the agent is suitable for topical administration. If theagent comprises soluble carrier with a plurality of fibrinogen bindingprecursors immobilised to each carrier, the agent may be suitable forintravenous administration.

The carrier or agent may be provided as a component of a kit. The kitmay further comprise a coagulation factor, a promoter of wound healing,or an antimicrobial agent. The coagulation factor, promoter of woundhealing, or antimicrobial agent may be a separate component of the kit,or immobilised to the carrier. In some preferred embodiments thecoagulation factor, promoter of wound healing, or antimicrobial agentmay be part of the fibrinogen binding precursor such that it is releasedwhen the fibrinogen binding precursor is converted to a fibrinogenbinding moiety.

A kit of the invention may be a compartmentalised kit in whichcomponents of the kit that are desired to be kept separate from oneanother are contained in separate compartments or containers. A kit ofthe invention may include instructions for using the kit components tocarry out a method of the invention.

Biogel or tissue adhesive of the invention may be topically administeredto a tissue site, for example to skin or mucosal tissue. Biogel ortissue adhesive of the invention may be used for haemostasis, as asealant, for localized drug delivery or for tissue engineering.

Biogel or tissue adhesive of the invention may be administered byforming the gel or adhesive before contacting the gel or adhesive withthe administration site, or by forming the biogel or tissue adhesive atthe administration site.

It has also been appreciated that an agent comprising soluble carrier,wherein a plurality of fibrinogen binding precursors each of which canbe converted to a fibrinogen binding moiety, are immobilised to eachcarrier may be administered intravenously, for example to controlbleeding or for drug delivery. A preferred intravenous formulation is a20% aqueous isotonic solution. In a preferred embodiment, the fibrinogenbinding precursors can be converted by thrombin.

There is further provided according to the invention a method ofcontrolling bleeding, which comprises intravenously administering anagent comprising soluble carrier, wherein a plurality of fibrinogenbinding precursors each of which can be converted to a fibrinogenbinding moiety, are immobilised to each carrier.

There is further provided according to the invention a method ofdelivering a drug to a subject, which comprises intravenouslyadministering an agent comprising soluble carrier to the subject,wherein a plurality of fibrinogen binding precursors each of which canbe converted to a fibrinogen binding moiety, are immobilised to eachcarrier, and wherein the carrier comprises a drug or a drug isimmobilised to the carrier.

Of course topical or intravenous formulations should be sterile.

Biogel or tissue adhesive of the invention may further comprise apromoter of wound healing, or an antimicrobial agent.

There is also provided according to the invention a method ofcontrolling bleeding, which comprises topically administering biogel ortissue adhesive of the invention to a bleeding site.

There is also provided according to the invention a method of treatingor sealing a wound, which comprises administering (preferably topically)a biogel or tissue adhesive of the invention to a wound site.

There is also provided according to the invention a biogel or tissueadhesive of the invention for use as a medicament.

There is further provided according to the invention use of a biogel ortissue adhesive of the invention in the manufacture of a medicament forcontrolling bleeding, or for treating or sealing a wound.

There is also provided according to the invention a biogel or tissueadhesive of the invention for controlling bleeding, or for treating orsealing a wound.

Use of a biogel or tissue adhesive of the invention may be topical use.Where the biogel or tissue adhesive is formed with soluble carrier andfibrinogen binding moieties converted from fibrinogen bindingprecursors, the use may be intravenous.

Preferred embodiments of the invention are now described by way ofexample only.

According to a first preferred embodiment of the invention, a pluralityof peptides, each comprising a fibrinogen binding sequence at theamino-terminal end (for example peptides of sequence GPRPGGGGGGC (SEQ IDNO: 24)) are linked at their carboxy-terminal ends to a carrier, whichis a soluble protein (such as albumin). A biogel is formed by contactingthe peptide-linked carrier with fibrinogen. The biogel may then beadministered topically as a dressing to a wound.

According to a second preferred embodiment of the invention, thepeptide-linked carrier of the first preferred embodiment and fibrinogenare mixed at a wound site to form a biogel in situ.

According to a third preferred embodiment of the invention, thepeptide-linked carrier of the first preferred embodiment is administeredto a wound site at which fibrinogen from host blood is present to form abiogel in situ.

According to a fourth preferred embodiment of the invention, a pluralityof peptides, each comprising a fibrinogen binding sequence coupled atits amino terminal end to a blocking peptide sequence (for examplepeptides of sequence LVPRGPRPGGGGGGC (SEQ ID NO: 25)), are linked attheir carboxy-terminal ends to a carrier, which is a soluble protein(such as albumin). The peptide-linked carrier can be combined withfibrinogen and then applied to a wound as a single mixture. Hostthrombin present at the site of the wound cleaves the peptides torelease the blocking peptides and expose the fibrinogen bindingsequence. The carrier with exposed fibrinogen binding sequence reactswith the fibrinogen to form a biogel in situ.

Alternatively, the peptide-linked carrier of the fourth preferredembodiment can be administered intravenously. Host thrombin at the siteof a wound cleaves the peptides to release the blocking peptides andexpose the fibrinogen binding sequence. The carrier with exposedfibrinogen binding sequences reacts with host fibrinogen to controlbleeding at the wound site.

The biogel of the four preferred embodiments described above may be atissue adhesive.

An insoluble carrier (for example an albumin microsphere) may be usedinstead of the soluble protein carrier in any of the above preferredembodiments (other than for intravenous administration).

Branched polyethylene glycol (PEG) or any other biocompatible polymermay be used instead of the soluble protein carrier in any of the abovepreferred embodiments.

A coagulation factor may be immobilised to the carrier in any of theabove preferred embodiments, providing that where fibrinogen bindingprecursors are immobilised to the carrier, the coagulation factor willnot convert the fibrinogen binding precursors to fibrinogen bindingmoieties.

The carrier may further comprise a promoter of wound healing (forexample, a growth factor such as platelet-derived growth factor) in anyof the above preferred embodiments.

Further preferred embodiments of the invention are described in thefollowing examples with reference to the accompanying drawings in which:

FIG. 1 which shows schematically: (a) an albumin carrier having aplurality of fibrinogen binding peptides immobilised to the carrier; (b)a fibrinogen molecule; and (c) a biogel in which the fibrinogenmolecules are linked together via the carriers by non-covalent bondsbetween the fibrinogen binding peptides and the fibrinogen molecules;

FIG. 2 shows a plot of the storage (squares) and loss modulus(triangles) against percentage strain on a mixture of 25 ul ofpeptide-modified HSA and fibrinogen at 32 mg/ml at 20° C. (A) and 37° C.(B);

FIG. 3 shows storage modulus (squares) and complex viscosity (triangles)versus frequency at 20° C. (with 1% strain amplitude), for 25 ul ofpeptide-modified HSA mixed with fibrinogen at 32 mg/ml; and

FIG. 4 shows a photograph of a fibrinogen solution before (a) and after(b) addition of peptide-modified HSA.

EXAMPLE 1 Formation of a Biogel or Tissue Adhesive Using Fibrinogen, andFibrinogen Binding Peptides Immobilised to a Carrier Comprising HumanSerum Albumin

Human serum albumin (HSA) was reacted with a 40-fold molar excess of thelinker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) at pH 7.4. The modified protein (HSA-SMCC) was purified and itwas determined that on average one mole of HSA was modified with 18moles of SMCC. Either GPRPGGGGGGC (B10; SEQ ID NO: 24) orLVPRGPRPGGGGGGC (TC-15; SEQ ID NO: 25) peptide was added to HSA-SMCC at1.5 mole excess in relation to maleimide moieties. The peptide-modifiedHSA protein was then purified from un-reacted peptide and stored at −80°C. at 7 mg/ml. Lyophilised human fibrinogen was solubilized in water at16 mg/ml as described by supplier (Scottish National Blood Transfusionservice).

Peptide-modified HSA (5 μl) was added to 0.3 ml of fibrinogen solution,an instant gel was observed for B10-HSA while for TC-15 no gel wasobserved. The exposed GPRP peptide sequence is known to bind to the “a”pocket within the carbonyl region of the two distal domains (D) offibrinogen (FIG. 1b ). It is believed that the HSA modified withmultiple exposed GPRP sequences (B10) acts as a branching point forpolymerisation of fibrinogen (FIG. 1c ). The gel is formed in theabsence of thrombin.

Methods for Examples 2-5 Synthesis of Peptide-Modified HSA

Human serum albumin (HSA) at 10 mg/ml was reacted with a 5, 10, 20 or40-fold molar excess of the linkersulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) at pH 7.4 with a total volume of 0.7 ml. The modifiedprotein (HSA-SMCC) was purified by a Zebra™ Desalt Spin Column (Pierce,Rockford, Ill.) and it was determined that on average one mole of HSAwas modified with 5, 8, 16 or 18 moles of Sulfo-SMCC, respectively. Todetermine the number of maleimide groups bound per HSA molecule, asample of 3 nmoles of HSA was incubated with a known amount of cysteine(100 nmoles) in buffer at room temperature for 30 min. The remainingcysteine was then reacted with 1 mmol of 5,5-dithiobis(2-nitrobenzoate)(DTNB) for 20 min and A₄₁₂ was measured. The maleimide level wascalculated by comparing the absorbance of the control and proteinsamples. Either GPRPGGGGGGC (B10; SEQ ID NO: 24) or LVPRGPRPGGGGGGC(TC-15; SEQ ID NO: 25) peptide was added to HSA-SMCC at 1.5 mole excessin relation to maleimide moieties. The peptide-modified HSA protein wasthen purified from un-reacted peptide using a Zebra™ Desalt Spin Column(Pierce, Rockford, Ill.) and stored at −80° C. at 7 mg/ml. Lyophilisedhuman fibrinogen (Scottish National Blood Transfusion Service,Edinburgh, Scotland) was solubilised in water at 8, 16, 32 and 64 mg/ml.The concentration of fibrinogen and albumin was determined from theabsorbance at 280 nm (A₂₈₀) using a conversion factor of 1% FibrinogenE₂₈₀=15 and 1% HSA E₂₈₀=13.8

Rheological Characterization

Dynamic oscillatory measurements were used to evaluate the viscoelasticproperties of the gels. The mixture was allowed to equilibrate at roomtemperature overnight and the gel was transferred to the lower plate ofa Physica MCR-501 (Anton Paar, Germany) rheometer with a cone and plateof 25 mm diameter and a 1° cone angle. Tests were performed at either 20or 37±° C. in a humidified atmosphere. Frequency sweeps were performedbetween 0.01 and 50 Hz at a constant strain amplitude of 1%. Therheological parameters examined were processed with the dedicated AntonPaar software provided with the rheometer.

Example 2 Influence of Both the Amount of Peptide-Modified HSA andFibrinogen Concentration on Gel Formation

The polymerisation of fibrinogen was initially monitored visually afterthe mixing of fibrinogen with HSA solutions. Peptide-modified HSAmodified with either 5, 8, 16 or 18 moles of fibrinogen binding peptidewas conjugated to one mole of HSA. A 25 ul sample of thepeptide-modified HSA or unmodified HSA was mixed with 0.2 ml offibrinogen at 8, 16, 32 or 64 mg/ml. The formation of a gel was visuallydetermined after 0.5 h at room temperature. For unmodified HSA or HSAmodified with 5 or 8 moles of peptide no gel formation was observed atall fibrinogen concentrations tested. Polymerisation was only observedat 32 or 64 mg/ml fibrinogen with peptide-modified HSA with 16 or 18moles of binding peptide.

FIG. 4 shows a photograph of the fibrinogen solution (0.2 ml 32 mg/mlfibrinogen) before (a) and after (b) the addition of peptide-modifiedHSA (25 μl, 18 moles peptide). The biogel formed according to theinvention is shown in (b).

Example 3 Influence of the Amount of Peptide-Modified HSA

To determine the effect of the amount of peptide-modified HSA andfibrinogen on the rheological properties of the mixtures, shear strengthmeasurements were determined with 25 or 50 ul of peptide-modified HSAwith fibrinogen at 32 and 64 mg/ml. Peptide-modified HSA that wasmodified with 16 moles of binding-peptide per mole of HSA was used forthis experiment. To ensure that the dynamic oscillatory experiments weremade within the linear viscoelastic strain limit of the gel a strainsweep was performed. FIG. 2 shows the strain sweeps at 20 and 37° C. for25 ul of peptide-modified HSA mixed with fibrinogen at 32 mg/ml. Bothstorage modulus (G′) and loss modulus (G″) appeared stable over therange and a constant strain of 1% was selected with in this linearviscoelastic strain range. Frequency sweeps demonstrated a G′ valuewhich was greater than G″ which is indicative of a cross-linked network(Table 1). Values shown in this Table are taken at a frequency of 1 Hz.Tan delta is the ratio of the loss modulus to the storage modulus. It istherefore a quantification of the elastic and viscous contributions,where a value above 1 is indicative of liquid like viscous behaviour andbelow 1 signifies elastic behaviour. Complex viscosity η*, defined ascomplex modulus G* divided by angular frequency (ω), were determinedunder the same conditions. It appears under these conditions that amountof both peptide-modified HSA and fibrinogen does not influence greatlythe mechanical strength of the gel.

TABLE 1 Fibrinogen (mg/ml) 32 64 Peptide-modified HSA (ul) 25 50 25 50Storage modulus (G′) 432 439 421 588 Complex viscosity (η*) 88 70 77 94Tan delta 0.22 0.11 0.24 0.2

A different batch of peptide-modified HSA with 18 peptides bound per HSA(25 ul) with 32 mg/ml fibrinogen showed G′ of 5200, complex viscosity of820 and Tan delta 0.105.

Example 4 Influence of Temperature on Rheological Properties

Investigations of the effect of temperature on a gel are important forapplications of the product when used at surgical body temperature orwhen applied topically.

We evaluated the mechanical strength of the gel at 20 and 37° C. andvalues given in Table 2 are taken at a frequency of 1 Hz. Increasing thetemperature of the gel generated from fibrinogen at 32 mg/ml and 25 uLof HSA modified with 16 moles of peptide resulted in both a decrease inthe G′ and complex viscosity (Table 2). At both 20 and 37° C. the tandelta was below 1, therefore a (non-covalent) cross-linked networkstructure is maintained at 37° C.

TABLE 2 Temperature (° C.) 20 37 Storage modulus (G′) 432 109 Complexviscosity (η*) 88 21 Tan delta 0.22 0.72

Example 5 Gel Formation in Human Plasma

Investigating whether the peptide-modified HSA could polymerisefibrinogen in human plasma is important for applications of the productas a surgical glue/adhesive. Peptide-modified HSA (75 ul) modified with18 moles of peptide was mixed with 0.8 ml of human pooled plasma.Rheology on the plasma derived gel was performed at 37° C. and gave a G′value of 6100 with a tan delta of 0.104. It is concluded from thisresult that peptide-modified HSA can polymerise fibrinogen in humanplasma resulting a in (non-covalent) cross-linked network structure at37° C.

1-42. (canceled)
 43. A soluble agent for formation of a biogel,comprising one or more soluble carriers to each of which are covalentlyimmobilized a plurality of fibrinogen binding moieties, wherein eachcarrier comprises at least one of i) a protein and ii) a biocompatiblepolymer, wherein each fibrinogen binding moiety is a peptide comprisingthe amino acid sequence NH2-G(P,H)RX- as set forth in SEQ ID NO: 15 atits amino terminal end, where X is any amino acid and either proline orhistidine is present at position (P,H) in said amino acid sequence, andwherein the agent, upon being contacted with a plurality of fibrinogenmolecules when thrombin is absent, is capable of forming a biogel whichcomprises fibrinogen molecules and a plurality of carriers such that (i)each of said plurality of fibrinogen molecules binds at least twofibrinogen moieties, and (ii) in the biogel, the fibrinogen moleculesare linked together via the carriers by non-covalent bonds between thefibrinogen binding moieties and the fibrinogen molecules.
 44. The agentof claim 43, wherein the carrier comprises albumin.
 45. The agent ofclaim 44, wherein the albumin of the carrier is human serum albumin. 46.The agent of claim 43, wherein the carrier comprises polyethyleneglycol.
 47. The agent of claim 43, wherein each fibrinogen bindingmoiety comprises the amino acid sequence NH₂-GPRP- as set forth in SEQID NO:16 at its amino terminal end.
 48. The agent of claim 43, whereineach fibrinogen binding moiety is 4-30 amino acid residues in length.49. The agent of claim 43, wherein each fibrinogen moiety is 4-10 aminoacid residues in length.
 50. The agent of claim 43, wherein eachfibrinogen binding moiety is immobilized to the carrier via anon-peptide spacer.
 51. The agent of claim 50, wherein the non-peptidespacer comprises polyethylene glycol.
 52. The agent of claim 43, whereineach carrier has on average at least five fibrinogen binding moietiesper carrier.
 53. The agent of claim 43 which is provided in a liquidformulation.
 54. The agent of claim 53, wherein the liquid formulationis a sterile formulation suitable for topical administration to asubject.
 55. The agent of claim 43, wherein the carrier comprisesalbumin, and wherein each fibrinogen binding moiety comprises the aminoacid sequence NH₂-GPRP- as set forth in SEQ ID NO:16 at its aminoterminal end.
 56. The agent of claim 43, wherein the carrier comprisesalbumin and wherein each fibrinogen binding moiety is 4-30 amino acidresidues in length.
 57. The agent of claim 43, wherein the carriercomprises albumin, wherein each fibrinogen binding moiety comprises theamino acid sequence NH₂-GPRP- as set forth in SEQ ID NO:16 at its aminoterminal end, and wherein each fibrinogen binding moiety is 4-30 aminoacid residues in length.
 58. The agent of claim 43, wherein the carriercomprises albumin, wherein each fibrinogen binding moiety comprises theamino acid sequence NH₂-GPRP- as set forth in SEQ ID NO:16 at its aminoterminal end, wherein each fibrinogen binding moiety is 4-30 amino acidresidues in length and wherein each fibrinogen binding moiety isimmobilized to the carrier via a non-peptide spacer.
 59. A biogel whichcomprises fibrinogen molecules and a plurality of carriers, wherein eachcarrier has a plurality of fibrinogen binding moieties covalentlyimmobilized to the carrier, and each molecule of fibrinogen is bound toat least two fibrinogen binding moieties, so that the fibrinogenmolecules are linked together via the carriers by non-covalent bondsbetween the fibrinogen binding moieties and the fibrinogen molecules,wherein each carrier comprises at least one of i) a protein and ii) abiocompatible polymer, and wherein each fibrinogen binding moiety is apeptide comprising the amino acid sequence NH2-G(P,H)RX- as set forth inSEQ ID NO: 15 at its amino terminal end.
 60. A method of forming abiogel which comprises contacting fibrinogen molecules with the agentfor formation of a biogel of claim
 43. 61. A method of controllingbleeding, which comprises topically administering the agent of claim 43to a bleeding site.
 62. A method of controlling bleeding, whichcomprises topically administering the biogel of claim 59 to a bleedingsite.
 63. A method of treating or sealing a wound, which comprisestopically administering the agent of claim 43 to a wound site.
 64. Amethod of treating or sealing a wound, which comprises topicallyadministering the biogel of claim 59 to a wound site.